The long-term OBJECTIVE of our work is to gain a deep mechanistic understanding of the fundamental process of how a simple epithelium forms by defining how cell-cell adhesion initiates actin remodeling and polarized cell organization. Our RATIONALE is that a simple epithelium is the fundamental building block of metazoans and some non-metazoans. It comprises a closed monolayer of quiescent, functionally polarized cells held together by cell-cell adhesion complexes and surrounded by the extracellular matrix (ECM). Our results from the current period showed that the mechanistic link between cell-cell adhesion and epithelial polarity is ancient and fundamental to understanding how a simple epithelium forms. However, the current inventory of proteins and pathways involved in cadherin-mediated adhesion is likely incomplete, and is complicated by the overlapping roles of many of those proteins/pathways in other adhesion systems and cell migration. Our STRATEGY is to exploit the results of a novel genome-wide RNAi screen for Ca++- and E-cadherin-dependent cell-cell adhesion in Drosophila S2 cells that excluded Ca++-independent cell-cell adhesion, integrin-based ECM adhesion and spreading, and cell migration. Based on a stringent screen and validation protocol, we have provisionally assigned ~100 confirmed hits to 6 intersecting regulatory hubs that contain many proteins not previously associated directly with cadherin function. OUR GOALS in the next period are: 1). systematically define the functions of signaling pathways involved in cadherin-mediated cell-cell adhesion and the organization of epithelial polarity across a range of scales. We will use a prioritized list of proteins from the DE-cad/S2 screen, together with the spatial map and time line of protein reorganization during cell-cell adhesion defined in our previous work; and 2). Examine signaling pathways involved in the response of cell-cell adhesion complexes to perturbation of epithelial homeostasis upon mechanical strain, which results in the re-entry of quiescent cells into the cell cycle through different pathways that are dependent on E-cadherin, beta-catenin and alpha-catenin. We anticipate that our results will provide new a mechanistic understanding of pathways that regulate epithelial morphogenesis and homeostasis.